Anti-PD-1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases

Abstract

Osteosarcoma (OS) pulmonary metastasis translates into poor patient survival. The implication of PD-1-PD-L1 pathway in the context of NK cells and/or macrophages in OS is unknown. We investigated the effect of anti-PD-1 in OS lung metastasis and the role of NK cells and/or macrophages in anti-PD-1 responses. A human LM7 OS mouse model was used. Immunohistochemistry for tissues (PD-L1, caspase-3, Ki-67, NK cells, macrophages), and Western blotting for OS lung tumors (p-Stat3, p-Erk1/2) was performed. NK and macrophages were assessed using flow cytometry. NK cell and macrophage depletion were conducted using anti-asialo GM1 and clodrosome, respectively. PD-L1 expression was observed in human OS cells and OS patient lung metastases. Anti-PD1 antibody led to a significant decrease in the number of OS lung metastases, enhanced tumor apoptosis, decreased tumor cell proliferation, and p-STAT-3/p-Erk1/2 signaling blockade in OS lung tumors. NK cells and macrophages in OS lung tumors expressed PD-1 and anti-PD1 increased NK cell and macrophage tumor infiltration. Increased numbers of antitumor M1 macrophages and decreased pro-inflammatory M2 macrophages were seen. NK depletion did not affect therapeutic effect of anti-PD-1, suggesting that NK cells were not directly involved. However, macrophage depletion significantly compromised anti-PD1 efficacy, confirming their role in efficacy of anti-PD-1 against OS lung metastasis. Our findings suggest that OS lung metastases regression by anti-PD1 can be attributed to activated tumor M1 macrophages and reduced M2 macrophages. Owing to the co-relation of M1 macrophages with OS patient outcome, we provide a novel mechanism of PD-1 blockade and a basis for future clinical trials for anti-PD-1 antibodies in OS.

Keywords: Immunotherapy; lung metastasis; macrophages; osteosarcoma; programmed death ligand-1.